The long-term goal of this study is to understand how an intracellular pathogen interacts with its host to establish a protected niche in which the pathogen can replicate. For the bacterial pathogen Listeria monocytogenes establishing and maintaining its intracellular niche, the host cytosol, requires precise spatial regulation of an essential virulence factor, listeriolysin O (LLO). LLO is a secreted pore-forming protein that mediates bacterial escape from the host vacuole to the host cytosol. Despite being continuously secreted by the bacterium, LLO is active only in the host vacuole. We have identified a cis-acting sequence in the amino terminus of LLO that is necessary to restrict the activity of LLO to the vacuole. Mutants that lack this sequence fail to correctly compartmentalize LLO activity, permeabilize the host plasma membrane in addition to the vacuolar membrane, and consequently destroy their intracellular niche. Importantly, these mutants are avirulent in vivo. The above sequence is rich in the amino acids proline (P), glutamate (E), serine (S), and threonine (T) and thus resembles eukaryotic PEST sequences. PEST sequences can target eukaryotic proteins for phosphorylation and/or degradation. We have shown that a mutant LLO lacking the PEST region accumulates to higher intracellular levels than the wild type protein suggesting that this region may influence intracellular stability of LLO. In addition, we have shown that LLO is phosphorylated in the host cytosol and that mutants lacking potential phospho-acceptor sites located within the PEST sequence also permeabilize the host plasma membrane and have decreased virulence. The specific goal of this proposal is to understand the mechanism by which LLO's PEST sequence regulates the protein's activity in the host cytosol. In Aim 1, we will identify specific residues within LLO's PEST sequence that are important for its function and which may represent contact sites for interacting host molecules. In Aim 2, we will define the pathway of LLO degradation and determine whether the PEST sequence affects this pathway. Lastly, in Aim 3, we will define the role of intracellular phosphorylation of LLO. Controlling when and where a virulence factor acts is critical for an intracellular pathogen to orchestrate a productive infection and cause disease. By defining the mechanism by which LLO activity is compartmentalized within a host cell, we will learn more about how intracellular pathogens can take advantage of host cell machinery to regulate the activity of key virulence factors that function within the host cytosol.